Method of Inducing And/Or Enhancing An Immune Response to Tumor Antigens

ABSTRACT

An improved method of inducing and/or enhancing an immune response to a tumor antigen is disclosed. The method involves administering the tumor antigen, nucleic acid coding therefor, vectors and/or cells comprising said nucleic acid, or vaccines comprising the aforementioned to a lymphatic site.

FIELD OF THE INVENTION

The present invention relates to methods for inducing and/or enhancingimmune responses to tumor antigens.

BACKGROUND OF THE INVENTION

Using immunological approaches to cancer therapy has been difficult astumor cells are self-derived and therefore not as immunogenic as inexogenous agents such as bacteria and viruses. As a result, theprospects of cancer immunotherapy rely upon the identification of tumorassociated antigens (“TAA”) which can be recognized by the immunesystem. Specifically, target antigens eliciting T cell-mediatedresponses are of critical interest. This comes from evidence thatcytotoxic T lymphocytes (CTLs) can induce tumor regression both inanimal models (Kast W. et al (1989) Cell 59:6035; Greendberg P. (1991)Adv. Immunol. 49:281) and in humans (Boon T. et al. (1994) Annu. Rev.Immunol. 12:337). To date, many tumor associated antigens have beenidentified. These include the antigens MAGE, BAGE, GAGE, RAGE, gp100,MART-1/Melan-A, tyrosinase, carcinoembryonic antigen (CEA) as well asmany others (Horig and Kaufman (1999) Clinical Immunology 92:211-223).Some of these tumor associated antigens are discussed below.

The first human tumor associated antigen characterized was identifiedfrom a melanoma. This antigen (originally designated MAGE 1) wasidentified using CTLs isolated from an HLA A1+ melanoma patient toscreen HLA A1 target cells transfected with tumor DNA (van der BruggenP. (1991) Science, 254:1643; these tumor associated antigens are nowdesignated MAGE-A1, MAGE-A2, etc). Interestingly, MAGE 1 was found tobelong to a family of at least 12 closely related genes located on the Xchromosome (de Plaen, E. et al. (1994) Immunogenetics 40:360). Thenucleic acid sequence of the 11 additional MAGE genes share 65-85%identity with that of MAGE-1 (de Smet, C. et al. (1994) Immunogenetics39:121). Both MAGE 1 and 3 are present in normal tissues, but expressedonly in the testis (de Plaen, E. et al. (1994) Supra; de Smet, C. et al.(1994) Supra; Takahashi, K. et al. (1995) Cancer Res. 55:3478; Chyomey,P. et al. (1995) Immunogenetics 43:97). These initial results havesubsequently been extended with the identification of new gene families(i.e. RAGE, BAGE, GAGE), all of which are typically not expressed innormal tissues (except testis) but expressed in a variety of tumortypes.

Human carcinoembryonic antigen (CEA) is a 180 kD glycoprotein expressedon the majority of colon, rectal, stomach and pancreatic tumors (Muaroet al. (1985) Cancer Res. 45:5769), some 50% of breast carcinomas(Steward et al. (1974) Cancer 33:1246) and 70% of lung carcinomas(Vincent, R. G. and Chu, T. M. (1978) J. Thor. Cardiovas. Surg. 66:320).CEA was first described as a cancer specific fetal antigen inadenocarcinoma of the human digestive tract in 1965 (Gold, P. andFreeman, S. O. (1965) Exp. Med. 121:439). Since that time, CEA has beencharacterized as a cell surface antigen produced in excess in nearly allsolid tumors of the human gastrointestinal tract. The gene for the humanCEA protein has been cloned (Oikawa et al (1987) Biochim. Biophys. Res.142:511-518; European Application No. EP 0346710). CEA is also expressedin fetal gut tissue and to a lesser extent on normal colon epithelium.The immunogenicity of CEA has been ambiguous, with several studiesreporting the presence of anti-CEA antibodies in patients (Gold et al.(1973) Nature New Biology 239:60; Pompecki. R. (1980) Eur. J. Cancer16:973; Ura at al. (1985) Cancer Lett. 25:283; Fuchs et al. (988) CancerImmunol. Immunother. 26:180) while other studies have not (LoGerfo etal. (1972) Int. J. Cancer 9:344; MacSween, J. M. (1975) Int. J. Cancer15:246; Chester K. A. and Begent, H. J. (1984) Clin. Exp. Immunol.58:685).

Gp100 is normally found in melanosomes and expressed in melanocytes,retinal cells, and other neural crest derivatives. The function of gp100is currently unknown. By mass spectrometry, three immunodominant HLA-A2binding gp100 epitopes have been identified: g9-154 (amino acids154-162), g9-209 (amino acids 209-217); and g9-280 (amino acids280-288). Notably, two of these epitopes (as peptides) have beensynthetically altered so as to induce a more vigorous immune response inthe original T cell clone: the threonine at position 2 in gp-209 waschanged to a methionine, and the alanine residue at position 9 in gp-280was changed to a valine. These changes increase the binding affinity ofthe epitope-peptides to the HLA-A2 molecule without changing theintrinsic natural epitopes recognized by the T cell receptor (TCR).Rosenberg and colleagues (NIH) have already successfully immunizedmelanoma patients with one of these modified peptides and have reportedachieving objective clinical responses in some patients.

Despite significant advances that have been made with respect toimmunological approaches to cancer treatment, there is still a need inthe art to improve cancer immunotherapies.

SUMMARY OF THE INVENTION

The present invention relates to improved methods for inducing and/orenhancing an immune response to a tumor antigen.

The present inventors have found that administering the tumor antigen ornucleic acid coding therefor directly into a lymphatic site (such as alymph node) induces and/or significantly enhances the immune response tothe tumor antigen and/or breaks tolerance to the tumor antigen, bothwhich have been a major challenge in previous methods of cancerimmunotherapy.

Accordingly, one aspect the present invention provides a method forinducing and/or enhancing an immune response in an animal to a tumorantigen comprising administering an effective amount of a tumor antigen,nucleic acid coding therefor, vector or cell comprising said nucleicacid, or vaccine comprising the aforementioned to a lymphatic site inthe animal.

In another aspect, the present invention provides a method for breakingimmune tolerance to a tumor antigen in an animal comprisingadministering an effective amount of a tumor antigen, nucleic acidcoding therefor, vector or cell comprising said nucleic acid, or vaccinecomprising the aforementioned to a lymphatic site in the animal.

Other features and advantages of the present invention will becomeapparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples while indicating preferred embodiments of the invention aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be described in relation to the drawings inwhich:

FIG. 1 is a bar graph showing the results of an IFN-γ-ELISPOT analysisof an animal receiving an intranodal injection of the tumor antigen.

FIG. 2 is a bar graph showing the results of an IFN-γ-ELISPOT analysisof an animal receiving an intranodal injection of the tumor antigen.

FIG. 3 is a bar graph showing the results of an IFN-γ-ELISPOT analysisof an animal receiving a subcutaneous injection of the tumor antigen.

FIG. 4 is a bar graph showing the results of an IFN-γ-ELISPOT analysisof an animal receiving a subcutaneous injection of the tumor antigen.

FIG. 5 is a graph showing the antibody response after a regiment ofintranodal (group 2) and subcutaneous (group 3) administration ofALVAC-modified gp100/modified gp100 peptide immunogens.

FIG. 6 is the nucleic acid sequence of a modified gp100M cDNA(SEQ.ID.NO.:109).

FIG. 7 is the deduced amino acid sequence of the modified gp100M protein(SEQ.ID.NO.:110).

FIG. 8 is the nucleic acid and amino acid sequence of a modified CEA(SEQ.ID.NOS.: 111 and 112).

DETAILED DESCRIPTION OF THE INVENTION

As hereinbefore mentioned, the present invention relates to an improvedmethod for inducing and/or enhancing the immune response to a tumorantigen. Accordingly, the present invention provides a method forinducing and/or enhancing an immune response in an animal to a tumorantigen comprising administering an effective amount of a tumor antigen,a nucleic acid sequence encoding a tumor antigen, a vector or cellcomprising the nucleic acid sequence, or a vaccine comprising the tumorantigen, the nucleic acid sequence encoding the tumor antigen, or avector comprising the nucleic acid sequence encoding the tumor antigento a lymphatic site in the animal.

The term “inducing and/or enhancing an immune response” means that themethod evokes and/or enhances any response of the animal's immunesystem.

“Immune response” is defined as any response of the immune system, forexample, of either a cell-mediated (i.e. cytotoxic T-lymphocytemediated) or humoral (i.e. antibody mediated) nature. These immuneresponses can be assessed by a number of in vivo or in vitro assays wellknown to one skilled in the art including, but not limited to, antibodyassays (for example ELISA assays) antigen specific cytotoxicity assays,production of cytokines (for example ELISPOT assays), regression oftumors expressing the tumor antigens, inhibition of cancer cellsexpressing the tumor antigens, etc.

The term “lymphatic site” means a site in the body that is associatedwith the lymphatic system including lymphatic organs, tissues, cells,nodes or glands such as spleen, thymus, tonsils, Peyer's patches, bonemarrow, lymphocytes, thoracic duct as well as all of the lymph nodes ofthe body.

The term “animal” as used herein includes all members of the animalkingdom and is preferably human.

The term “effective amount” as used herein means an amount effective, atdosages and for periods of time necessary to achieve the desiredresults.

The term “tumor antigen” as used herein includes both tumor associatedantigens (TAAs) and tumor specific antigens (TSAs). A tumor associatedantigen means an antigen that is expressed on the surface of a tumorcell in higher amounts than is observed on normal cells or an antigenthat is expressed on normal cells during fetal development. A tumorspecific antigen is an antigen that is unique to tumor cells and is notexpressed on normal cells. The term tumor antigen includes TAAs or TSAsthat have been already identified and those that have yet to beidentified and includes fragments, epitopes and any and allmodifications to the tumor antigens.

The tumor associated antigen can be any tumor associated antigenincluding, but not limited to, gp100 (Kawakami et al., J. Immunol.154:3961-3968 (1995); Cox et al., Science, 264:716-719 (1994)),MART—1/Melan A (Kawakami et al., J. Exp. Med., 180:347-352 (1994);Castelli et al., J. Exp. Med., 181:363-368 (1995)), gp75 (TRP-1) (Wanget al., J. Exp. Med., 186:1131-1140 (1996)), and Tyrosinase (Wolfel etal., Eur. J. Immunol., 24:759-764 (1994); Topalian et al., J. Exp. Med.,183:1965-1971 (1996) melanoma proteoglycan (Hellstrom et al., J.Immunol., 130:1467-1 (1983); Ross et al., Arch. Biochem Biophys.,225:370-383 (1983)); amor-specific, widely shared antigens, for example:antigens of MAGE family, for example, MAGE-1, 2, 3, 4, 6, and 12 (Vander Bruggen et al., Science, 254:1643-1647 (1991); Rogner et al.,Genomics, 29:729-731 (1995)), antigens of BAGE family (Boel et al.,Immunity, 2:167-175 (1995)), antigens of GAGE family, for example,GAGE-1,2 (Van den Eynde et al., J. Exp. Med., 182:689-698 (1995)),antigens of RAGE family, for example, RAGE-1 (Gaugler et al.,Immunogenetics, 44:323-330 (1996)), N-acetylglucosaminyltransferase-V(Guilloux et al., J. Exp. Med., 183:1173-1183 (1996)), and p15 (Robbinset al., J. Immunol. 154:5944-5950 (1995)); tumor specific mutatedantigens; mutated β-catenin (Robbins et al., J. Exp. Med., 183:1185-1192(1996)), mutated MUM-1 (Coulie et al., Proc. Natl. Aced. Sci. USA,92:7976-7980 (1995)), and mutated cyclin dependent kinases-4 (CDK4)(Wolfel et al., Science, 269:1281-1284 (1995)); mutated oncogeneproducts: p21 ras (Fossum et al., Int. J. Cancer, 56:40-45 (1994)),BCR-abl (Bocchia et al., Blood, 85:2680-2684 (1995)), p53 (Theobald etal., Proc. Natl. Aced. Sci. USA, 92:11993-11997 (1995)), and p185HER2/neu (Fisk et al. J. Exp. Med., 181:2109-2117 (1995)); Peoples etal., Proc. Natl. Aced. Sci., USA, 92:432-436 (1995)); mutated epidermalgrowth factor receptor (EGFR) (Fujimoto et al., Eur J. Gynecol. Oncol.,16:40-47 (1995)); Harris et al., Breast Cancer Res. Treat, 29:1-2(1994)); carcinoembryonic antigens (CEA) (Kwong et al., J. Natl. CancerInst., 85:982-990 (1995)); carcinoma associated mutated mucins, forexample, MUC-1 gene products (Jerome et al., J. Immunol., 151:1654-1662(1993), Ioannides et al., J. Immunol. 151:36933703 (1993), Takahashi etal., J. Immunol., 153:2102-2109 (1994)); EBNA gene products of EBV, forexample, EBNA-1 gene product (Rickinson et al., Cancer Surveys, 13:53-80(1992)); E7, E6 proteins of human papillomavirus (Ressing et al., J.Immunol, 154:5934-5943 (1995)); prostate specific antigens (PSA) (Sue etal., The Prostate, 30:73-78 (1997)); prostate specific membrane antigen(PSMA) (Israeli, et al., Cancer Res., 54; 1807-1811 (1994)); PCTA-1 (Sueet al., Proc. Natl. Acad. Sci. USA, 93:7252-7257 (1996)), idiotypicepitopes or antigens, for example, immunoglobulin idiotypes or T cellreceptor idiotypes, (Chen et al., J. Immunol., 153:4775-4787 (1994);Syrengelas et al., Nat. Med., 2:1038-1040 (1996)); KSA (U.S. Pat. No.5,348,887); NY-ESO-1 (WO 98/14464).

Also included are modified tumor antigens and/or epitope/peptidesderived therefrom (both unmodified and modified). Examples include, butare not limited to, modified and unmodified epitope/peptides derivedfrom gp100 (WO 98/02598; WO 95/29193, WO 97/34613; WO 98/33810; CEA (WO99/19478; S. Zaremba et al. (1997) Cancer Research 57:4570-7; K. T.Tsang et al. (1995) J. Int. Cancer Inst. 87:982-90); MART-1 (WO98/58951, WO 98/02538; D. Valmeri et al. (2000) J. Immunol.164:1125-31); p53 (M. Eura et al. (2000) Clinical Cancer Research6:979-86); TRP-1 and TRP-2 (WO 97/29195); tyrosinase (WO 96/21734; WO97/11669; WO 97/34613; WO 98/33810; WO 95/23234; WO 97/26535); KSA (WO97/15597); PSA (WO 96/40754); NY-ESO 1 (WO 99/18206); HER2/neu (U.S.Pat. No. 5,869,445); MAGE family related (L. Heidecker et al. (2000) J.Immunol. 164:6041-5; WO 95/04542; WO 95/25530; WO 95/25739; WO 96/26214;WO 97/31017; WO 98/10780).

In a preferred embodiment, the tumor-associated antigen is gp100, amodified gp100 or a fragment thereof. In particular, the inventors haveprepared a modified gp100 peptide termed gp100M which has the nucleicacid sequence shown in FIG. 6 (SEQ.ID.NO.:109) and the deduced aminoacid sequence shown in FIG. 7 (SEQ.ID.NO.:110). The inventors have shownthat the intranodal injection of a recombinant avipox virus comprising anucleic acid coding for fragments of the modified gp100 (comprisingmodified epitopes 209(2M) (IMDQVPFSY, SEQ.ID.NO.:1) and 290(9V)(YLEPGPVTV, SEQ.ID.NO.:2)) followed by modified epitope/peptide boostsinduced both a humoral and cell mediated response that was several timeshigher than when the same antigens were administered subcutaneously. Theexperimental details and results are discussed in Example 1.

In another embodiment, the tumor-associated antigen is carcinoembryonicantigen (CEA), a modified CEA or a fragment thereof. The nucleic acidsequence of a modified CEA antigen is shown in FIG. 8 andSEQ.ID.NO.:111. The corresponding amino acid sequence is shown in FIG. 8and SEQ.ID.NO.:112. Preferably, the modified CEA antigen comprises thesequence YLSGADLNL, SEQ.ID.NO.:113.

Additional embodiments of the invention encompass nucleic acid sequencescomprising sequences encoding the tumor antigens and fragments ormodified forms thereof as hereinbefore described. The term “nucleic acidsequence” refers to a sequence of nucleotide or nucleoside monomersconsisting of naturally occurring bases, sugars and intersugar(backbone) linkages. The term also includes modified or substitutedsequences comprising non-naturally occurring monomers or portionsthereof, which function similarly. The nucleic acid sequences of thepresent invention may be ribonucleic (RNA) or deoxyribonucleic acids(DNA) and may contain naturally occurring bases including adenine,guanine, cytosine, thymidine and uracil. The sequences may also containmodified bases such as xanthine, hypoxanthine, 2-aminoadenine, 6-methyl,2-propyl, and other alkyl adenines, 5-halo uracil, 5-halo cytosine,6-aza uracil, 6-aza cytosine and 6-aza thymine, pseudo uracil,4-thiouracil, 8-halo adenine, 8-amino adenine, 8-thiol adenine,8-thio-alkyl adenines, 8-hydroxyl adenine and other 8-substitutedadenines, 8-halo guanines, 8-amino guanine, 8-thiol guanine, 8-thioalkylguanines, 8-hydroxyl guanine and other 8-substituted guanines, other azaand deaza uracils, thymidines, cytosines, adenines, or guanines,5-trifluoromethyl uracil and 5-trifluoro cytosine.

The nucleic acid sequences encoding the tumor antigens of the inventioninclude, but are not limited to, viral nucleic acid(s), plasmid(s),bacterial DNA, naked/free DNA and RNA. The nucleic acids encompass bothsingle and double stranded forms. As such, these nucleic acids comprisethe relevant base sequences coding for the aforementioned tumorantigens. For purposes of definitiveness, the “relevant base sequencescoding for the aforementioned polypeptides” further encompasscomplementary nucleic acid sequences. As such, embodiments of theinvention encompass nucleic acid sequences per se encoding for theaforementioned tumor antigens, or recombinant nucleic acids into whichhas been inserted said nucleic acids coding for tumor antigens (asdescribed below).

Bacterial DNA useful in recombinant nucleic acid embodiments of theinvention are known to those of ordinary skill in the art. Sources ofbacterial DNA include, for example, Shigella, Salmonella, Vibriocholerae, Lactobacillus, Bacille Calmette Guérin (BCG), andStreptococcus. In bacterial DNA embodiments of the invention, nucleicacid of the invention may be inserted into the bacterial genome, canremain in a free state, or be carried on a plasmid.

Viral recombinant nucleic acid embodiments of the invention may bederived from a poxvirus or other virus such as adenovirus or alphavirus.Preferably the viral nucleic acid is incapable of integration inrecipient animal cells. The elements for expression from said nucleicacid may include a promoter suitable for expression in recipient animalcells.

Specific vial recombinant nucleic acid embodiments of the inventionencompass (but are not limited to) poxviral, alphaviral, and adenoviralnucleic acid. Poxviral nucleic acid may be selected from the groupconsisting of avipox, orthopox, and suipox nucleic acid. Particularembodiments encompass poxviral nucleic acid selected from vaccinia,fowlpox, canary pox and swinepox; specific examples include TROVAC,NYVAC, ALVAC, MVA, Wyeth and Poxvac-TC (described in more detail below).

It is further contemplated that recombinant nucleic acids of thisinvention may further comprise nucleic acid sequences encoding at leastone member chosen from the group consisting of cytokines, lymphokines,and co-stimulatory molecules. Examples include (but are not limited to)interleukin 2, interleukin 12, interleukin 6, interferon gamma, tumornecrosis factor Alpha, GM-CSF, B7.1, B7.2, ICAM-1, LFA-3, and Cd72.

Standard techniques of molecular biology for preparing and purifyingnucleic acids well known to those skilled in the art can be used in thepreparation of the recombinant nucleic acid aspects of the invention(for example, as taught in Current Protocols in Molecular Biology, F. M.Ausubel et al. (Eds.), John Wiley and Sons, Inc, N.Y., U.S.A. (1998),Chpts. 1, 2 and 4; Molecular Cloning: A Laboratory Manual (2^(nd) Ed.),J. Sambrook, E. F. Fritsch and T. Maniatis (Eds.). Cold Spring HarborLaboratory Press, N.Y., U.S.A. (1989), Chpts. 1, 2, 3 and 7).

Aspects of this invention further encompass vectors comprising theaforementioned nucleic acids. In certain embodiments, said vectors maybe recombinant viruses or bacteria (as described below).

Adenovirus vectors and methods for their construction have beendescribed (e.g. U.S. Pat. Nos. 5,994,132, 5,932,210, 6,057,158 andPublished PCT Applications WO 9817783, WO 9744475, WO 9961034, WO9950292, WO 9927101, WO 9720575, WO 9640955, WO 9630534-all of which areherein incorporated by reference). Alphavirus vectors have also beendescribed in the art and can be used in embodiments of this invention(e.g. U.S. Pat. Nos. 5,792,462, 5,739,026, 5,843,723, 5,789,245, andPublished PCT Applications WO 9210578, WO 9527044, WO 9531565, WO9815636-all of which are herein incorporated by reference), as havelentivirus vectors (e.g. U.S. Pat. Nos. 6,013,516, 5,994,136 andPublished PCT Applications WO 9817816, WO 9712622, WO 9817815, WO9839463, WO 9846083, WO 9915641, WO 9919501, WO 9930742, WO 9931251, WO9851810, WO 0000600-all of which are herein incorporated by reference).Poxvirus vectors that can be used include, for example, avipox, orthopoxor suipox poxvirus (as described in U.S. Pat. Nos. 5,364,773, 4,603,112,5,762,938, 5,378,457, 5,494,807, 5,505,941, 5,756,103, 5,833,975 and5,990,091—all of which are herein incorporated by reference). Poxvirusvectors comprising a nucleic acid coding for a tumor antigen can beobtained by homologous recombination as is known to one skilled in theart. As such, the nucleic acid coding for the tumor antigen is insertedinto the viral genome under appropriate conditions for expression inmammalian cells (as described below).

In one embodiment of the invention the poxvirus vector is ALVAC (1) orALVAC (2) (both of which have been derived from canarypox virus). ALVAC(1) (or ALVAC (2)) does not productively replicate in non-avian hosts, acharacteristic thought to improve its safety profile. ALVAC (1) is anattenuated canarypox virus-based vector that was a plaque-clonedderivative of the licensed canarypox vaccine, Kanapox (Tartaglia et al.(1992) Virology 188:217; U.S. Pat. Nos. 5,505,941, 5,756,103 and5,833,975-all of which are incorporated herein by reference). ALVAC (1)has some general properties which are the same as some generalproperties of Kanapox. ALVAC-based recombinant viruses expressingextrinsic immunogens have also been demonstrated efficacious as vaccinevectors (Tartaglia et al., In AIDS Research Reviews (vol. 3) Koff W.,Wong-Staol F. and Kenedy R. C. (eds.), Marcel Dekker NY, pp. 361-378(1993a); Tartaglia, J. et al. (1993b) J. Virol. 67:2370). For instance,mice immunized with an ALVAC (1) recombinant expressing the rabies virusglycoprotein were protected from lethal challenge with rabies virus(Tartaglia, J. et al., (1992) supra) demonstrating the potential forALVAC (1) as a vaccine vector. ALVAC-based recombinants have also provenefficacious in dogs challenged with canine distemper virus (Taylor, J.et al., (1992) Virology 187:321) and rabies virus (Perkus, M. E. et al.,In Combined Vaccines and Simultaneous Administration: Current Issues andPerspective, Annals of the New York Academy of Sciences (1994)), in catschallenged with feline leukemia virus (Tartaglia, J. et al., (1993b)supra), and in horses challenged with equine influenza virus (Taylor, J.et al., In Proceedings of the Third International Symposium on AvianInfluenza, Univ. of Wisconsin-Madison, Madison, Wis., pp. 331-335(1993)).

ALVAC (2) is a second-generation ALVAC vector in which vacciniatranscription elements E3L and K3L have been inserted within the C6locus (U.S. Pat. No. 5,990,091, incorporated herein by reference). TheE3L. encodes a protein capable of specifically binding to dsRNA. TheK3L. ORF has significant homology to E1F-2. Within ALVAC (2) the E3L.gene is under the transcriptional control of its natural promoter,whereas K3L has been placed under the control of the early/late vaccineH6 promoter. The E3L and K3L genes act to inhibit PKR activity in cellsinfected with ALVAC (II) allowing enhancement of the level andpersistence of foreign gene expression.

Additional viral vectors encompass natural host-restricted poxviruses.Fowlpox virus (FPV) is the prototypic virus of the Avipox genus of thePoxvirus family. Replication of avipox viruses is limited to avianspecies (Matthews, R. E. F. (1982) Intervirology 17:42) and there are noreports in the literature of avipox virus causing a productive infectionin any non-avian species including man. This host restriction providesan inherent safety barrier to transmission of the virus to other speciesand makes use of avipox virus based vectors in veterinary and humanapplications an attractive proposition.

FPV has been used advantageously as a vector expressing immunogens frompoultry pathogens. The hemagglutinin protein of a virulent avianinfluenza virus was expressed in an FPV recombinant. After inoculationof the recombinant into chickens and turkeys, an immune response wasinduced which was protective against either a homologous or aheterologous virulent influenza virus challenge (Taylor, J. et al.(1988) Vaccine 6: 504). FPV recombinants expressing the surfaceglycoproteins of Newcastle Disease Virus have also been developed(Taylor, J. et al. (1990) J. Virol. 64; 1441; Edbauer, C. at al. (1990)Virology 179:901; U.S. Pat. No. 5,766,599-incorporated herein byreference).

A highly attenuated strain of vaccinia, designated MVA, has also beenused as a vector for poxvirus-based vaccines. Use of MVA is described inU.S. Pat. No. 5,185,146.

Other attenuated poxvirus vectors have been prepared via geneticmodification to wild type strains of vaccinia. The NYVAC vector, forexample, is derived by deletion of specific virulence and host-rangegenes from the Copenhagen strain of vaccinia (Tartaglia, J. at al.(1992), supra; U.S. Pat. Nos. 5,364,773 and 5,494,807-incorporatedherein by reference) and has proven useful as a recombinant vector ineliciting a protective immune response against expressed foreignantigens.

Recombinant viruses can be constructed by processes known to thoseskilled in the art (for example, as previously described for vacciniaand avipox viruses; U.S. Pat. Nos. 4,769,330; 4,722,848; 4,603,112;5,110,587; and 5,174,993-all of which are incorporated herein byreference).

In further embodiments of the invention, live and/or attenuated bacteriamay also be used as vectors. For example, non-toxicogenic Vibriocholerae mutant strains may be useful as bacterial vectors inembodiments of this invention; as described in U.S. Pat. No. 4,882,278(disclosing a strain in which a substantial amount of the codingsequence of each of the two ctxA alleles has been deleted so that nofunctional cholera toxin is produced), WO 92111354 (strain in which theirgA locus is inactivated by mutation; this mutation can be combined ina single strain with ctxA mutations), and WO 94/1533 (deletion mutantlacking functional ctxA and attRS1 DNA sequences). These strains can begenetically engineered to express heterologous antigens, as described inWO 94/19482. (All of the aforementioned issued patent/patentapplications are incorporated herein by reference.)

Attenuated Salmonella typhimurium strains, genetically engineered forrecombinant expression of heterologous antigens and their use as oralimmunogens are described, for example, in WO 92/11361.

As noted, those skilled in the art will readily recognize that otherbacterial strains useful as bacterial vectors in embodiments of thisinvention include (but are not limited to) Shigella flexneri,Streptococcus gordonii, and Bacille Calmette Guerin (as described in WO88/6626, WO 90/0594, WO 91/13157, WO 92/1796, and WO 92/21376; all ofwhich are incorporated herein by reference). In bacterial vectorembodiments of this invention, a nucleic acid coding for a tumor antigenmay be inserted into the bacterial genome, can remain in a free state,or be carried on a plasmid.

It is further contemplated that the invention encompasses vectors whichcomprise nucleic acids coding for at least one member from the groupconsisting of cytokines, lymphokines and immunostimulatory molecules.Said nucleic acid sequences can be contiguous with sequences coding forthe tumor antigen or encoded on distinct nucleic acids.

Cells comprising the aforementioned tumor antigens, nucleic acids codingtherefor, and/or vectors encompass further embodiments of the invention.These cells encompass any potential cell into which the aforementionedtumor antigen, nucleic acid, and/or vector might be introduced and/ortransfected and/or infected or example, bacteria, COS cells, Vero cells,chick embryo fibroblasts, tumor cells, antigen presenting cells,dendritic cells, etc.). The choice of process for the introductionand/or transfection and/or infection into cells is dependant upon theintrinsic nature of the introduced agent (i.e. free DNA, plasmid,recombinant virus), as will be known to one skilled in the art (forexample, as taught in Current Protocols in Molecular Biology, F. M.Ausubel et al. (Eds.), John Wiley and Sons, Inc., N.Y, U.S.A. (1998),Chpt. 9; Molecular Cloning: A Laboratory Manuel (2nd Ed.), J. Sambrook,E. F. Fritsch and T. Maniatis (Eds.), Cold Spring Harbor LaboratoryPress. N.Y., U.S.A. (1989), Chpts. 1, 2, 3 and 16).

Further embodiments of the invention encompass vaccines comprising thetumor antigens and/or nucleic acids coding therefor and/or vectorsand/or cells previously described.

The vaccine of the invention comprising the tumor antigen may be amultivalent vaccine and additionally contain several peptides, epitopesor fragments of a particular tumor antigen or contain peptides relatedto other tumor antigens and/or infectious agents in a prophylacticallyor therapeutically effective manner. Multivalent vaccines againstcancers may contain a number of individual TAA's, or immunogenicfragments thereof, alone or in combinations which are effective tomodulate an immune response to cancer.

A vaccine of the invention may contain a nucleic acid molecule encodinga tumor antigen of the invention. Such vaccines are referred to asnucleic acid vaccines but are also termed genetic vaccines,polynucleotide vaccines or DNA vaccines, all of which are within thescope of the present invention. In such an embodiment, the tumor antigenis produced in vivo in the host animal. Additional embodiments if theinvention encompass vectors (i.e. bacteria, recombinant viruses)comprising the aforementioned nucleic acids.

The present invention also contemplates mixtures of the tumor antigens,nucleic acids coding therefor, vectors comprising said nucleic acids,cells and/or vaccines comprising the aforementioned, and at least onemember selected from the group consisting of cytokines, lymphokines,immunostimulatory molecules, and nucleic acids coding therefor.Additional embodiments of this invention further encompasspharmaceutical compositions comprising the aforementioned tumorantigens, nucleic acids coding therefor, vectors, cells, vaccines ormixtures for administration to subjects in a biologically compatibleform suitable for administration in vivo. By “biologically compatibleform suitable for administration in vivo” is meant a form of thesubstance to be administered in which any toxic effects are outweighedby the therapeutic effects. Administration of a therapeutically activeamount of the pharmaceutical compositions of the present invention, oran “effective amount”, is defined as an amount effective at dosages andfor periods of time, necessary to achieve the desired result ofeliciting an immune response in an animal. A therapeutically effectiveamount of a substance may vary according to factors such as the diseasestate/health, age, sex, and weight of the recipient, and the inherentability of the particular tumor antigen, nucleic acid coding therefor,vector, cell, or vaccine to elicit a desired immune response. Dosageregime may be adjusted to provide the optimum therapeutic response. Forexample, several divided doses may be administered daily, or at periodicintervals, and/or the dose may be proportionally reduced as indicated bythe exigencies of circumstances.

The pharmaceutical compositions described herein can be prepared by perse known methods for the preparation of pharmaceutically acceptablecompositions which can be administered to animals such that an effectivequantity of the active substance (i.e. tumor antigen, nucleic acid,recombinant virus, vaccine) is combined in a mixture with apharmaceutically acceptable vehicle. Suitable vehicles are described,for example, in “Handbook of Pharmaceutical Additives” (compiled byMichael and Irene Ash, Gower Publishing Limited, Aldershot, England(1995)). On this basis, the compositions include, albeit notexclusively, solutions of the substances in association with one or morepharmaceutically acceptable vehicles or diluents, and may be containedin buffered solutions with a suitable pH and/or be iso-osmotic withphysiological fluids. In this regard, reference can be made to U.S. Pat.No. 5,843,456 These compositions may further comprise an adjuvant (asdescribed below).

Further embodiments of the invention encompass methods of inhibiting atumor antigen expressing cancer cell in a patient comprisingadministering to said patient an effective amount of a tumor antigen,nucleic acid coding therefor, vector, cell, or vaccine of the invention.Patients with solid tumors expressing tumor antigens include (but arenot limited to) those suffering from colon cancer, lung cancer, pancreascancer, endometrial cancer, breast cancer, thyroid cancer, melanoma,oral cancer, laryngeal cancer, seminoma, hepatocellular cancer, bileduct cancer, squamous cell carcinoma, and prostate cancer. As such,methods of treating patients with cancer per se encompassing theaforementioned methods of inducing an immune response and/or inhibitinga tumor antigen expressing cell are contemplated aspects/embodiments ofthe invention.

As mentioned previously, an animal may be immunized with a tumorantigen, nucleic acid coding therefore, vector, cell or vaccine of theinvention by administering the aforementioned to a lymphatic site. Theadministration can be achieved in a single dose or repeated atintervals. The appropriate dosage is dependant on various parametersunderstood by the skilled artisans, such as the immunogen itself (i.e.polypeptide vs. nucleic acid (and more specifically type thereof)), theroute of administration and the condition of the animal to be vaccinated(weight, age and the like).

As previously noted, nucleic acids (in particular plasmids and/orfree/naked DNA and/or RNA coding for the tumor antigen of the invention)can be administered to an animal for purposes of inducing/eliciting animmune response (for example, U.S. Pat. No. 5,589,466; McDonnell andAskari, NEJM 334:42-45 (1996); Kowalczyk and Ertl, Cell Mol. Life Sci.55:751-770 (1999)). Typically, this nucleic acid is a form that isunable to replicate in the target animal's cell and unable to integratein said animal's genome. The DNA/RNA molecule encoding the tumor antigenis also typically placed under the control of a promoter suitable forexpression in the animal's cell. The promoter can function ubiquitouslyor tissue-specifically. Examples of non-tissue specific promotersinclude the early Cytomegalovirus (CMV) promoter (described in U.S. Pat.No. 4,168,062) and the Rous Sarcoma Virus promoter. The desmin promoteris tissue-specific and drives expression in muscle cells. Moregenerally, useful vectors have been described (i.e., WO 94/21797).

For administration of nucleic acids coding for a tumor antigen, saidnucleic acid can encode a precursor or mature form of thepolypeptide/protein. When it encodes a precursor form, the precursorform can be homologous or heterologous. In the latter case, a eucaryoticleader sequence can be used, such as the leader sequence of thetissue-type plasminogen factor (tPA).

For use as an immunogen, a nucleic acid of the invention can beformulated according to various methods known to a skilled artisan.First, a nucleic acid can be used in a naked/free form, free of anydelivery vehicles (such as anionic liposomes, cationic lipids,microparticles, (e.g., gold microparticles), precipitating agents (e.g.,calcium phosphate) or any other transfection-facilitating agent. In thiscase the nucleic acid can be simply diluted in a physiologicallyacceptable solution (such as sterile saline or sterile buffered saline)with or without a carrier. When present, the carrier preferably isisotonic, hypotonic, or weakly hypertonic, and has a relatively lowionic strength (such as provided by a sucrose solution (e.g., a solutioncontaining 20% sucrose)).

Alternatively, a nucleic acid can be associated with agents that assistin cellular uptake. It can be, i.e., (i) complemented with a chemicalagent that modifies the cellular permeability (such as bupivacaine; see,for example, WO 94/16737), (ii) encapsulated into liposomes, or (iii)associated with cationic lipids or silica, gold, or tungstenmicroparticles.

Cationic lipids are well known in the art and are commonly used for genedelivery. Such lipids include Lipofectin (also known as DOTMA(N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride), DOTAP(1,2-bis(oleyloxy)-3-(trimethylammonio) propane), DRAB(dimethyldioctadecyl-ammonium bromide), DOGS (dioctadecylamidologlycylspermine) and cholesterol derivatives such as DC-Choi (3beta-(N-(N′,N′-dimethyl aminomethane)-carbamoyl) cholesterol). Adescription of these cationic lipids can be found in EP 187702, WO90/11092, U.S. Pat. No. 5,283,185, WO 91/15501, WO 95/26356, and U.S.Pat. No. 5,527,928. Cationic lipids for gene delivery are preferablyused in association with a neutral lipid such as DOPE (dioleylphosphatidylethanolamine) as, for example, described in WO 90/11092.

Other transfection-facilitating compounds can be added to a formulationcontaining cationic liposomes. A number of them are described in, forexample, WO 93/18759, WO 93/19768, WO 94/25608, and WO 95/2397. Theyinclude, for example, spermine derivatives useful for facilitating thetransport of DNA through the nuclear membrane (see, for example, WO93/18759) and membrane-permeabilizing compounds such as GALA,Gramicidine S, and cationic bile salts (see, for example, WO 93/19768).

Gold or tungsten microparticles can also be used for nucleic aciddelivery (as described in WO 91/359 and WO 93/17706). In this case, themicroparticle-coated polynucleotides can be injected via intradermal orintraepidermal routes using a needleless injection device (“gene gun”;such as those described, for example, in U.S. Pat. No. 4,945,050, U.S.Pat. No. 5,015,580, and WO 94/24263).

Anionic and neutral liposomes are also well-known in the art (see, forexample, Liposomes: A Practical Approach, RPC New Ed, IRL Press (1990),for a detailed description of methods for making liposomes) and areuseful for delivering a large range of products, including nucleicacids.

Particular embodiments of the aforementioned methods (i.e. toinduce/elicit immune responses) encompass prime-boost protocols for theadministration of immunogens of the invention. More specifically, theseprotocols encompass (but are not limited to) a “priming” step with aparticular/distinct form of immunogen (i.e. nucleic acid (for example,plasmid, bacterial/viral/free or naked)) coding for tumor antigen, orvector (i.e. recombinant virus, bacteria) comprising said nucleic acid)followed by at least one “boosting” step encompassing the administrationof an alternate (i.e. distinct from that used to “prime”) form of thetumor antigen (i.e. protein or fragment thereof (for example,epitope/peptide), nucleic acid coding for the tumor antigen (or fragmentthereof), or vector comprising said nucleic acid). Examples of“prime-boost” methodologies are known to those skilled in the art (astaught, for example, in PCT published applications WO 98/58956, WO98/56919, WO 97/39771). One advantage of said protocols is the potentialto circumvent the problem of generating neutralizing immune responses tovectors per se (i.e. recombinant viruses) wherein isinserted/incorporated nucleic acids encoding the immunogen or fragmentsthereof (see for example, R. M. Conry et al. (2000) Clin. Cancer Res.6:34-41).

As is well known to those of ordinary skill in the art, the ability ofan immunogen to induce/elicit an immune response can be improved if,regardless of administration formulation (i.e. recombinant virus,nucleic acid, polypeptide), said immunogen is co-administered with anadjuvant. Adjuvants are described and discussed in “Vaccine Design—theSubunit and Adjuvant Approach” (edited by Powell and Newman, PlenumPress, New York. U.S.A., pp. 61-79 and 141-228 (1995)). Adjuvantstypically enhance the immunogenicity of an immunogen but are notnecessarily immunogenic in and of themselves. Adjuvants may act byretaining the immunogen locally near the site of administration toproduce a depot effect facilitating a slow, sustained release ofimmunizing agent to cells of the immune system. Adjuvants can alsoattract cells of the immune system to an immunogen depot and stimulatesuch cells to elicit immune responses. As such, embodiments of thisinvention encompass compositions further comprising adjuvants.

Desirable characteristics of ideal adjuvants include:

-   1) lack of toxicity;-   2) ability to stimulate a long-lasting immune response;-   3) simplicity of manufacture and stability in long-term storage;-   4) ability to elicit both cellular and humoral responses to antigens    administered by various routes, if required;-   5) synergy with other adjuvants;-   6) capability of selectively interacting with populations of antigen    presenting cells (APC);-   7) ability to specifically elicit appropriate TH1 or TH2    cell-specific immune responses; and-   8) ability to selectively increase appropriate antibody isotype    levels for example, IgA) against antigens/immunogens.

However, many adjuvants are toxic and can cause undesirable sideeffects, thus making them unsuitable for use in humans and many animals.For example, some adjuvants may induce granulomas, acute and chronicinflammations (i.e. Freund's complete adjuvant (FCA)), cytolysis (i.e.saponins and pluronic polymers) and pyrogenicity, arthritis and anterioruveitis (i.e. muramyl dipeptide (MDP) and lipopolysaccharide (LPS)).Indeed, only aluminum hydroxide and aluminum phosphate (collectivelycommonly referred to as alum) are routinely used as adjuvants in humanand veterinary vaccines. The efficacy of alum in increasing antibodyresponses to diphtheria and tetanus toxoids is well established.Notwithstanding, it does have limitations. For example, alum isineffective for influenza vaccination and inconsistently elicits a cellmediated immune response with other immunogens. The antibodies elicitedby alum-adjuvanted antigens are mainly of the IgG1 isotype in the mouse,which may not be optimal for protection in vaccination contexts.

Adjuvants may be characterized as “intrinsic” or “extrinsic”. Intrinsicadjuvants (such as lipopolysaccharides) are integral and normalcomponents of agents which in themselves are used as vaccines (i.e.killed or attenuated bacteria). Extrinsic adjuvants are typicallynonintegral immunomodulators generally linked to antigens in anoncovalent manner, and are formulated to enhance the host immuneresponse.

In embodiments of the invention, adjuvants can be at least one memberchosen from the group consisting of cytokines, lymphokines, andco-stimulatory molecules. Examples include (but are not limited to)interleukin 2, interleukin 12, interleukin 6, interferon gamma, tumornecrosis factor alpha, GM-CSF, B7.1, B7.2, ICAM-1, LFA-3, and CD72.Particular embodiments specifically encompass the use of GM-CSF as anadjuvant (as taught, for example, in U.S. Pat. Nos. 5,679,356,5,904,920, 5,637,483, 5,759,535, 5,254,534, European Patent ApplicationEP 211684, and published PCT document WO 97/28816—all of which areherein incorporated by reference).

A variety of potent extrinsic adjuvants have been described. Theseinclude (but are not limited to) saponins complexed to membrane proteinantigens (immune stimulating complexes), pluronic polymers with mineraloil, killed mycobacteria and mineral oil, Freund's complete adjuvant,bacterial products such as muramyl dipeptide (MOP) andlipopolysaccharide (LPS), as well as lipid A, and liposomes.

The use of saponins per se as adjuvants is also well known(Lacaille-Dubois, M. and Wagner, H. (1996) Phytomedicine 2:363). Forexample, Quil A (derived from the bark of the South American treeQuillaja Saponaria Molina) and fractions thereof has been extensivelydescribed (i.e. U.S. Pat. No. 5,057,540; Kensil, C. R. (1996) Crit RevTher Drug Carrier Syst. 12:1; and European Patent EP 362279). Thehaemolytic saponins QS21 and QS17 (HPLC purified fractions of Quil A)have been described as potent systemic adjuvants (U.S. Pat. No.5,057,540; European Patent EP 362279). Also described in thesereferences is the use of QS7 (a non-haemolytic fraction of Quil-A) whichacts as a potent adjuvant for systemic vaccines. Use of QS21 is furtherdescribed in Kensil et al. ((1991) J. Immunol. 146:431). Combinations ofQS21 and polysorbate or cyclodextrin are also known (WO 9910008).Particulate adjuvant systems comprising fractions of Quil A (such asQS21 and QS7) are described in WO 9633739 and WO 9611711.

Another preferred adjuvant/immunostimulant is an immunostimulatoryoligonucleotide containing unmethylated CpG dinucleotides (“CpG”). CpGis an abbreviation for cytosine-guanosine dinucleotide motifs present inDNA. CpG is known in the art as being an adjuvant when administered byboth systemic and mucosal routes (WO 9602555; European Patent EP 468520;Davies et al., (1998) J. Immunol, 160:87; McCluskie and Davis (1998) J.Immunol. 161:4463). In a number of studies, synthetic oligonucleotidesderived from BCG gene sequences have also been shown to be capable ofinducing immunostimulatory effects (both in vitro and in vivo; Krieg,(1995) Nature 374:546). Detailed analyses of immunostimulatoryoligonucleotide sequences has demonstrated that the CG motif must be ina certain sequence context, and that such sequences are common inbacterial DNA but are rare in vertebrate DNA. (For example, theimmunostimulatory sequence is often: purine, purine, C, G, pyrimidine,pyrimidine, wherein the CG motif is not methylated; however otherunmethylated CpG sequences are known to be immunostimulatory and as suchmay also be used in the present invention.) As will be evident to one ofnormal skill in the art, said CG motifs/sequences can be incorporatedinto nucleic acids of the invention per se, or reside on distinctnucleic acids.

A variety of other adjuvants are taught in the art, and as such areencompassed by embodiments of this invention. U.S. Pat. No. 4,855,283granted to Lockhoff et al. (incorporated herein by reference) teachesglycolipid analogues and their use as adjuvants. These includeN-glycosylamides, N-glycosylureas and N-glycosylcarbamates, each ofwhich is substituted in the sugar residue by an amino acid, asimmune-modulators or adjuvants. Furthermore, Lockhoff et al. ((1991)Chem. Int. Ed. Engl. 30:1611) have reported that N-glycolipid analogsdisplaying structural similarities to the naturally-occurringglycolipids (such as glycophospholipids and glycoglycerolipids) are alsocapable of eliciting strong immune responses in both herpes simplexvirus vaccine and pseudorabies virus vaccine.

U.S. Pat. No. 4,258,029 granted to Moloney (incorporated herein byreference) teaches that octadecyl tyrosine hydrochloride (OTH) functionsas an adjuvant when complexed with tetanus toxoid and formalininactivated type I, II and III poliomyelitis virus vaccine. Nixon-Georgeet al. ((1990) J. Immunol. 14:4798) have also reported that octadecylesters of aromatic amino acids complexed with a recombinant hepatitis Bsurface antigen enhanced the host immune responses against hepatitis Bvirus.

Adjuvant compounds may also be chosen from the polymers of acrylic ormethacrylic acid and the copolymers of maleic anhydride and alkenylderivative. Adjuvant compounds are the polymers of acrylic ormethacrylic acid which are cross-linked, especially with polyalkenylethers of sugars or polyalcohols. These compounds are known by the termcarbomer (Pharmeuropa Vol. 8, No. 2, June 1996). Preferably, a solutionof adjuvant according to the invention, especially of carbomer, isprepared in distilled water, preferably in the presence of sodiumchloride, the solution obtained being at acidic pH. This stock solutionis diluted by adding it to the desired quantity (for obtaining thedesired final concentration), or a substantial part thereof, of watercharged with NaCl, preferably physiological saline (NaCL 9 g/l) all atonce in several portions with concomitant or subsequent neutralization(pH 7.3 to 7.4), preferably with NaOH. This solution at physiological pHwill be used as it is for mixing with the immunizing agent said mixturebeing amenable to storage in the freeze-dried, liquid or frozen form.

Persons skilled in the art can also refer to U.S. Pat. No. 2,909,462(incorporated herein by reference) which describes adjuvantsencompassing acrylic polymers cross-linked with a polyhydroxylatedcompound having at least 3 hydroxyl groups (preferably not more than 8),the hydrogen atoms of the at least three hydroxyls being replaced byunsaturated aliphatic radicals having at least 2 carbon atoms. Thepreferred radicals are those containing from 2 to 4 carbon atoms (e.g.vinyls, allyls and other ethylenically unsaturated groups). Theunsaturated radicals may themselves contain other substituents, such asmethyl. The products sold under the name Carbopol (BF Goodrich, Ohio,USA) are particularly appropriate. They are cross-linked with allylsucrose or with allyl pentaerythritol. Among them, there may bementioned Carbopol (for example, 974P, 934P and 971P). Among thecopolymers of maleic anhydride and alkenyl derivative, the copolymersEMA (Monsanto; which are copolymers of maleic anhydride and ethylene,linear or cross-linked, (for example cross-linked with divinyl ether))are preferred. Reference may be made to J. Fields et al. ((1960) Nature186: 778) for a further description of these chemicals (incorporated(herein by reference).

In further aspects of this invention, adjuvants useful for parenteraladministration of immunizing agent include aluminum compounds (such asaluminum hydroxide, aluminum phosphate, and aluminum hydroxy phosphate;but might also be a salt of calcium, iron or zinc, or may be aninsoluble suspension of acylated tyrosine, or acylated sugars,cationically or anionically derivatised polysaccharides, orpolyphosphazenes). The antigen can be precipitated with, or adsorbedonto, the aluminum compound according to standard protocols well knownto those skilled in the art.

Other adjuvants encompassed by embodiments of this invention includelipid A (in particular 3-de-O-acylated monophosphoryl lipid A (3D-MPL).3D-MPL is a well known adjuvant manufactured by Ribi Immunochem,Montana. It is often supplied chemically as a mixture of 3-de-O-acylatedmonophosphoryl lipid A with 4, 5, or 6 acylated chains. It can beprepared by the methods taught in GB 21222048. A preferred form of3D-MPL is in the form of a particulate formulation having a particlesize less than 0.2 μm in diameter (European Patent EP 689454).

Adjuvants for mucosal immunization may include bacterial toxins (e.g.,the cholera toxin (CT), the E. coli heat-labile toxin (LT), theClostridium difficile toxin A and the pertussis toxin (PT), orcombinations, subunits, toxoids, or mutants thereof). For example, apurified preparation of native cholera toxin subunit B (CTB) can be ofuse. Fragments, homologs, derivatives, and fusion to any of these toxinsare also suitable, provided that they retain adjuvant activity. A mutanthaving reduced toxicity may be used. Mutants have been described (e.g.,in WO 95/17211 (Arg-7-Lys CT mutant), WO 96/6627 (Arg-192-Gly LTmutant), and WO 95/34323 (Arg-9-Lys and Glu-129-Gly PT mutant)).Additional LT mutants include, for example Ser-63-Lys, Ala-69-Gly,Glu-110-Asp, and Glu-112-Asp mutants. Other adjuvants (such as abacterial monophosphoryl lipid A (MPLA)) of various sources (e.g., E.coli, Salmonella minnesota, Salmonella typhimurium, or Shigellaflexneri) can also be used in the mucosal administration of immunizingagents.

Adjuvants useful for both mucosal and parenteral immunization includepolyphosphazene (for example, WO 95/2415), DC-chol (3b-(N-(N′,N′-dimethyl aminomethane)-carbamoyl) cholesterol (for example,U.S. Pat. No. 5,283,185 and WO 96/14831) and QS-21 (for example, WO88/9336).

Adjuvants/immunostimulants as described herein may be formulatedtogether with carriers, such as for example liposomes, oil in wateremulsions, and/or metallic salts including aluminum salts (such asaluminum hydroxide). For example, 3D-MPL may be formulated with aluminumhydroxide (as discussed in EP 689454) or oil in water emulsions (asdiscussed in WO 9517210); QS21 may be advantageously formulated withcholesterol containing liposomes (as discussed in WO 9633739), in oilwater emulsions (as discussed in WO 9517210) or alum (as discussed in WO9815287). When formulated into vaccines, immunostimulatoryoligonucleotides (i.e. CpGs) are generally administered in free solutiontogether with free antigen (as discussed in WO 9602555; McCluskie andDavis (1998) Supra), covalently conjugated to an antigen (as discussedin WO 9816247), or formulated with a carrier such as aluminum hydroxideor alum (as discussed in Davies et al. Supra; Brazolot-Millan et al(1998) Proc. Natl. Acad. Sci. 95:15553).

Combinations of adjuvants/immunostimulants are also within the scope ofthis invention. For example, a combination of a monophosphoryl lipid Aand a saponin derivative (as described in WO 9400153, WO 9517210, WO9633739, WO 9856414, WO 9912565, WO 9911214) can be used, or moreparticularly the combination of QS21 and 30-MPL (as described in WO9400153). A combination of an immunostimulatory oligonucleotide and asaponin (such as QS21), or a combination of monophosphoryl lipid A(preferably 3D-MPL) in combination with an aluminum salt also form apotent adjuvant for use in the present invention.

The following non-limiting example is illustrative of the presentinvention:

Examples Example 1

This example compares the intranodal injection with subcutaneousinjection of a representative tumor antigen (modified gp100).

Methods and Experimental Design Test System

Cynomolgus monkeys (Macaca fascicularis) purpose bred animals. Supplier:Siconbrec “Simian Conservation Breeding & Research Center Inc.”, FemaBuilding, 44 Gil Puyat Avenue Makati, Metro Manila, Philippines. Numberof animals in the study: 12 (6 males and 6 females).Age at initiation of treatment: 26 to 38 months.

-   -   Body weight range at initiation of treatment (day −1):    -   males: 1.73 to 2.34 kg    -   females: 1.71 to 2.65 kg.

Animal Husbandry

-   -   Housing: one air-conditioned room;    -   temperature: 19 to 25° C. (target range),    -   relative humidity: >40%    -   air changes: minimum 8 air changes per hour,    -   lighting cycle: 12 hours light (artificial)/12 hours dark.    -   Caging: animals were housed singly in stainless steel mesh cages        (approximately 540×810×760 mm).    -   Diet: expanded complete commercial primate diet (Mazuri diet,        Special Diet Services Ltd., Witham, Essex, CM8, 3AD, Great        Britain) analyzed for chemical and bacterial contaminants.        Quantity distributed: 100 g diet/animal/day.        In addition, animals received fruit daily (apple or banana)        Animals were fasted for at least 16 hours before blood sampling        for clinical laboratory investigations and before necropsy.    -   Water: drinking water ad libitum (via bottles).    -   Contaminants: no known contaminants were present in diet or        water at levels which might have interfered with achieving the        objective of the study.

Pre-Treatment Procedures

-   -   Animal health procedure: all animals received a clinical        examination for ill-health on arrival and a veterinary clinical        examination during the acclimatization period.    -   Acclimatization period: at least 3 weeks between animal arrival        and start of treatment.

Experimental Design

Allocation to treatment groups was performed during the acclimatizationperiod using a random allocation procedure based on body weight classes.

-   -   Animals were assigned to the treatment groups shown in Table 1.        The dose levels administered were shown in Table 2.

Administration of the Test/Control Articles Group 1 and 2 Animals

-   -   Method of administration: injection in the left inguinal lymph        node. Animals were lightly anaesthetized before each        administration by an intramuscular injection of ketmine        hydrochloride (Imalgene® 500—Merial, Lyon, France). The same        lymph node was injected on each occasion (left side). Each        injection was followed by a local disinfection with iodine        (Vétédine®—Vétoquinol, Lure, France).

Group 3

-   -   Route: subcutaneous.    -   Method of administration: bolus injection using a sterile        syringe and needle introduced subcutaneously. Four injection        sites were used followed by a local disinfection with iodine        (Vétédine®-Vétoquinol, Lure, France). Animals were also lightly        anaesthetized before each administration by an intramuscular        injection of ketamine hydrochloride (Imalgene® 500—Merial, Lyon,        France) in order to be under the same conditions as groups 1 and        2 animals.        Four injection sites in the dorsal cervical/interscapular        regions were used as shown in Table 3.

ELISPOT Analysis

An ELISPOT assay was used in order to assess the cell mediated immuneresponse generated in the monkeys in the various treatment groups. Inparticular, an ELISPOT IFNγ assay was used in order to measure IFNγproduction from T lymphocytes obtained from the monkeys in response togp100 antigens.

Materials and Methods

Plates: MILLIPORE Multiscreen HA plate/MAHA S45.10 (96 wells).Capture antibodies: MABTECH monoclonal anti-IFNγ antibodies/G-Z4 1mg/mL.Detection antibodies: MABTECH monoclonal anti-IFNγantibodies/7-86-1-biotin 1 mg/mL.Enzyme: SIGMA, Extravidin-PA conjuate/E2636Substrate: BIORAD, NBT/BCIP—Alkaline phosphatase conjugate substratekit/ref: 170-64 32.

Coating

Place 100 μL per well of capture antibodies at 1 μg/mL diluted at 1/1000in carbonate bicarbonate buffer 0.1M pH 9.6 into the multiwell plate.Incubate overnight at 4° C. Wash 4 times in 1×PBS.

Saturation

Place 200 μL per well of RPMI supplemented with 10% FCS, non essentialamino acids, pyruvate, Hepes buffer and Peni-Strepto. Incubate 2 hoursat 37° C.

Test

Cells from the immunized animals are tested against (a) medium alone;(b) pooled peptides at a concentration of 1 mg/mL; and (c) a nonspecific stimulus (PMA-lono). The pooled peptides used in this Exampleto stimulate IFN-γ production were derived from gp100 and areillustrated in Tables 4 to 7. The final volume of each sample is 200 μL.Incubate 20 hours at 37° C.Wash 4 times in 1×PBS and 0.05% Tween 20.

Detection

Place 100 μL per well of detection antibodies at 1 μg/mL diluted in1/1000 1×PBS, 1% BSA and 0.05% Tween 20. Incubate 2 hours at roomtemperature. Wash 4 times in 1×PBS and 0.05% Tween 20.

Reaction

Place 100 μL per well of Extravidin-PA conjugate diluted 1/6000 in1×PBS, 1% BSA and 0.05% Tween 20. Incubate 45 minutes at roomtemperature.Wash 4 times in 1×PBS and 0.05% Tween 20.

Substrate Addition

Place 100 μL per well of substrate previously prepared. For example, for1 plate, prepare: 9.6 μL of distilled water, 0.4 mL of 25× buffer, 0.1mL of solution A (NBT) and 0.1 mL of solution. B (BCIP). Incubate 30-45minutes at room temperature. Wash in distilled water. Dry and transferto a plastic film. The number of spots are counted using a Zeiss imageanalyzer. Each spot corresponds to an individual IFN-γ secreting T cell.

Results

The animals that tested positive on the ELISPOT analysis are shown inFIGS. 1-4. Overall, the results demonstrate that of the animals tested,2 out of 2 (i.e. 100%) of the animals that received the intranodaladministration of the gp100 antigen, and 2 out of 4 (i.e. 50%) of theannals that received the subcutaneous administration of the gp100antigen had a positive cell mediated immune response.

ELISA Analysis

The ELISA was performed utilizing standard methodology known in the art.Briefly, the human gp100 (“hgp100”; produced in Baculovirus) was dilutedin coating buffer (carbonate-bicarbonate, pH9.6) and added to 96 wellsat 0.5 ug/well. Plates were placed at 4° C. overnight. Plates were thenwashed and blocking buffer (phosphate buffered saline/0.5% Tween 20/1.0%BSA, pH7.2) was added for 2 hours at 37° C. The plates were then washedand the sera was diluted in dilution buffer (phosphate bufferedsaline/0.5% Tween 20/0.1 BSA, pH72). For this study, monkey sera wasdiluted to 1:800 and “7” serial 3 fold dilutions were done for eachsample tested. The human sera controls were diluted to 1:50 in dilutionbuffer and “7” serial 2 fold dilutions were performed. Each dilution wasdone in duplicate. The plates were incubated a further 2 hours at 37° C.The plates were washed and the horse radish peroxidase (HRP)-conjugatedanti-human secondary antibody (anti-human Ig whole antibody from sheep(Amersham Life Science, NA933)) diluted 1:100 in dilution buffer wasadded to the wells and incubated for 1 hour at 37° C. The plates werewashed and OPD (o-phenylenediamine dihydrochloride) substrate with H₂O₂in substrate buffer (50 mM phosphate/25 mM citrate, pH 7.2) was added tothe wells. For a kinetics ELISA, the plate was read repeatedly (2 minuteintervals for 15 minutes) unstopped (without “stop” buffer). Plates wereread at 450 nm.

Results

The results of the above experiment are presented in Table 8 and in FIG.5. The animals of group 2 received intranodal injections ofALVAC(2)-gp100(mod) followed by boosts with the modified gp100 peptides209(2M) and 290(9V); the animals in group 3 received a subcutaneousinjection of the ALVAC(2) construct followed by peptide boosts; theanimals in group 1 received intranodal injections of saline as acontrol.

As can be seen from FIG. 5, intranodal injection of the antigens induceda humoral response that was much greater than when the antigen wasinjected subcutaneously.

In summary, the results of this Example demonstrate that intranodalinjection of a tumor antigen induces both a humoral and cell mediatedresponse that is much greater than when the tumor antigen is injected bythe conventional subcutaneous route of administration.

While the present invention has been described with reference to whatare presently considered to be the preferred examples, it is to beunderstood that the invention is not limited to the disclosed examples.To the contrary, the invention is intended to cover variousmodifications and equivalent arrangements included within the spirit andscope of the appended claims.

All publications, patents and patent applications are hereinincorporated by reference in their entirety to the same extent as ifeach individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety.

TABLE 1 Number Group Route of Treatment days and compound of Numberadministration administered Animals 1 Intranodal Saline (NaCl 0.9%):days 28, 42, 56 4 Then 70, 71, 72, 73, 74 Then 84, 85, 86, 87 and 88 2Intranodal ALVAC(2) - gp100 mod: days 28, 42, 4 56 *mgp100 peptides:days 70, 71, 72, 73, 74 Then 84, 85, 86, 87 and 88 3 Subcutaneous Saline(NaCl 0.9%): day 1 4 ALVAC(2) - gp100 mod: days 28, 42, 56 *mgp100peptides: days 70 and 84 *209(2M)-IMDQVPFSY; 290(9V) YLEPGPVTV Group 1animals (control) received the control article (saline for injection(NaCl 0.9%)). Group 3 animals received the control article (saline forinjection (NaCl 0.9%)) on day 1 only.

TABLE 2 Dose volume Group (ml/ Number Dose level administration) 1Saline (NaCl 0.9%): 0 0.250 2 Dose: 0.25 × 10^(7.4) CCID 50 0.250 ALVAC(2) - gp100 mod: 0.25 10^(7.4) CCID50 Dose: 200 μg (Total) of peptidesIMDQVPFSY 0.2 (209(2M)), and YLEPGPVTV (290(9V)) (100 μg each) 3 Saline(NaCl 0.9%) 0.250 ALVAC(2) - gp100 mod: 0.25 10^(7.4) CCID 50 0.250Dose: 200 μg (Total) of peptides IMDQVPFSY 0.2 (209(2M)), and YLEPGPVTV(290(9V)) (100 μg each)

TABLE 3 Days Sites used 1 and 28 lower left 42 upper left 56 upper right70 lower left 84 lower right

TABLE 4 Peptide Pool #1 Peptide Sequence SEQ. ID. NO. 1329HLAVIGALLAVGATK SEQ. ID. NO. 3 1330 GALLAVGATKVPRNQ SEQ. ID. NO. 4 1331VGATKVPRNQDWLGV SEQ. ID. NO. 5 1332 VPRNQDWLGVSRQLR SEQ. ID. NO. 6 1333DWLGVSRQLRTKAWN SEQ. ID. NO. 7 1334 SRQLRTKAWNRQLYP SEQ. ID. NO. 8 1335TKAWNRQLYPEWTEA SEQ. ID. NO. 9 1336 RQLYPEWTEAQRLDC SEQ. ID. NO. 10 1337EWTEAQRLDCWRGGQ SEQ. ID. NO. 11 1338 QRLDCWRGGQVSLKV SEQ. ID. NO. 121339 WRGGQVSLKVSNDGP SEQ. ID. NO. 13 1340 VSLKVSNDGPTLIGASEQ. ID. NO. 14 1344 IALNFPGSQKVLPDG SEQ. ID. NO. 15 1345PGSQKVLPDGQVIWV SEQ. ID. NO. 16 1346 VLPDGQVIWVNNTII SEQ. ID. NO. 171347 QVIWVNNTIINGSQV SEQ. ID. NO. 18 1348 NNTIINGSQVWGGQPSEQ. ID. NO. 19 1349 NGSQVWGGQPVYPQE SEQ. ID. NO. 20 1350WGGQPVYPQETDDAC SEQ. ID. NO. 21 1351 VYPQETDDACIFPDG SEQ. ID. NO. 221352 TDDACIFPDGGPCPS SEQ. ID. NO. 23 1353 IFPDGGPCPSGSWSQSEQ. ID. NO. 24 1355 GSWSQKRSFVYVWKT SEQ. ID. NO. 25 1356KRSFVYVWKTWGQYW SEQ. ID. NO. 26 1357 YVWKTWGQYWQVLGG SEQ. ID. NO. 271358 WGQYWQVLGGPVSGL SEQ. ID. NO. 28 1359 QVLGGPVSGLSIGTGSEQ. ID. NO. 29

TABLE 5 Peptide Pool #2 Peptide Sequence SEQ. ID. NO. 1360PVSGLSIGTGRAMLG SEQ. ID. NO. 30 1361 SIGRGRAMLGTHTME SEQ. ID. NO. 311362 RAMLGTHTMEVTVYH SEQ. ID. NO. 32 1363 THTMEVTVYHRRGSRSEQ. ID. NO. 33 1364 VTVYHRRGSRSYVPL SEQ. ID. NO. 34 1365RRGSRSYVPLAHSSS SEQ. ID. NO. 35 1366 SYVPLAHSSSAFTIT SEQ. ID. NO. 361368 AFTITDQVPFSVSVS SEQ. ID. NO. 37 1369 DQVPFSVSVSQLRALSEQ. ID. NO. 38 1370 SVSVSQLRALDGGNK SEQ. ID. NO. 39 1372DGGNKHFLRNQPLTF SEQ. ID. NO. 40 1373 HFLRNQPLTFALQLH SEQ. ID. NO. 411374 QPLTFALQLHDPSGY SEQ. ID. NO. 42 1375 ALQLHDPSGYLAEADSEQ. ID. NO. 43 1379 DFGDSSGTLISRALV SEQ. ID. NO. 44 1380STGLISRALVVTHTY SEQ. ID. NO. 45 1381 SRALVVTHTYLEPGP SEQ. ID. NO. 461382 VTHTYLEPGPVTAQV SEQ. ID. NO. 47 1383 LEPGPVTAQVVLQAASEQ. ID. NO. 48 1384 VTAQVVLQAAIPLTS SEQ. ID. NO. 49 1385VLQAAIPLTSCGSSP SEQ. ID. NO. 50 1386 IPLTSCGSSPVPGTT SEQ. ID. NO. 511388 VPGTTDGHRPTAEAP SEQ. ID. NO. 52 1389 DGHRPTAEAPNTTAGSEQ. ID. NO. 53 1390 TAEAPNTTAGQVPTT SEQ. ID. NO. 54 1392QVPTTEVVGTTPGQA SEQ. ID. NO. 55 1393 EVVGTTPGQAPTAEP SEQ. ID. NO. 56

TABLE 6 Peptide Pool #3 Peptide Sequence SEQ. ID. NO. 1394TPGQAPTAEPSGTTS SEQ. ID. NO. 57 1395 PTAEPSGTTSVQVPT SEQ. ID. NO. 581396 SGTTSVQVPTTEVIS SEQ. ID. NO. 59 1397 VQVPTTEVISTAPVQSEQ. ID. NO. 60 1398 TEVISTAPVQMPTAE SEQ. ID. NO. 61 1399TAPVQMPTAESTGMT SEQ. ID. NO. 62 1400 MPTAESTGMTPEKVP SEQ. ID. NO. 631401 STGMTPEKVPVSEVM SEQ. ID. NO. 64 1402 PEKVPVSEVMGTTLASEQ. ID. NO. 65 1403 VSEVMGTTLAEMSTP SEQ. ID. NO. 66 1404GTTLAEMSTPEATGM SEQ. ID. NO. 67 1405 EMSTPEATGMTPAEV SEQ. ID. NO. 681408 SIVVLSGTTAAQVTT SEQ. ID. NO. 69 1409 SGTTAAQVTTTEWVESEQ. ID. NO. 70 1410 AQVTTTEWVETTARE SEQ. ID. NO. 71 1411TEWVETTARELPIPE SEQ. ID. NO. 72 1412 TTARELPIPEPEGPD SEQ. ID. NO. 731413 LPIPEPEGPDASSIM SEQ. ID. NO. 74 1414 PEGPDASSIMSTESISEQ. ID. NO. 75 1415 ASSIMSTESITGSLG SEQ. ID. NO. 76 1416STESITGSLGPLLDG SEQ. ID. NO. 77 1417 TGSLGPLLDGTATLR SEQ. ID. NO. 781418 PLLDGTATLRLVKRQ SEQ. ID. NO. 79 1419 TATLRLVKRQVPLDCSEQ. ID. NO. 80 1420 LVKRQVPLDCVLYRY SEQ. ID. NO. 81 1421VPLDCVLYRYGSFSV SEQ. ID. NO. 82 1422 VLYRYGSFSVTLDIV SEQ. ID. NO. 83

TABLE 7 Peptide Pool #4 Peptide Sequence SEQ. ID. NO. 1424TLDIVQGIESAEILQ SEQ. ID. NO. 84 1425 QGIESAEILQAVPSG SEQ. ID. NO. 851426 AEILQAVPSGEGDAF SEQ. ID. NO. 86 1427 AVPSGEGDAFELTVSSEQ. ID. NO. 87 1428 EGDAFELTVSCQGGL SEQ. ID. NO. 88 1429ELTVSCQGGLPKEAC SEQ. ID. NO. 89 1430 CQGGLPKEACMEISS SEQ. ID. NO. 901431 PKEACMEISSPGCQP SEQ. ID. NO. 91 1432 MEISSPGCQPPAQRLSEQ. ID. NO. 92 1434 PAQRLCQPVLPSPAC SEQ. ID. NO. 93 1435CQPVLPSPACQLVLH SEQ. ID. NO. 94 1436 PSPACQLVLHQILKG SEQ. ID. NO. 951437 QLVLHQILKGGSGTY SEQ. ID. NO. 96 1441 LADTNSLAVVSTQLISEQ. ID. NO. 97 1442 SLAVVSTQLIMPGQE SEQ. ID. NO. 98 1443STQLIMPGQEAGLGQ SEQ. ID. NO. 99 1444 MPGQEAGLGQVPLIV SEQ. ID. NO. 1001445 AGLGQVPLIVGILLV SEQ. ID. NO. 101 1448 LMAVVLASLIYRRRLSEQ. ID. NO. 102 1450 YRRRLMKQDFSVPQL SEQ. ID. NO. 103 1451MKQDFSVPQLPHSSS SEQ. ID. NO. 104 1452 SVPQLPHSSSHWLRL SEQ. ID. NO. 1051453 PHSSSHWLRLPRIFC SEQ. ID. NO. 106 1454 HWLRLPRIFCSCPIGSEQ. ID. NO. 107 1455 PRIFCSCPIGENSPL SEQ. ID. NO. 108

TABLE 8 DAY (mOD/min) Monkey # 0 57 68 96 1 3 5 2 2 2 4 6 12 10 3 7 6 108 4 7 6 8 8 5 5 9 20 15 6 11 8 10 12 7 11 23 51 30 8 7 30 70 22 9 1 7 53 10 2 6 6 4 11 3 7 14 8 12 6 9 15 6

1-19. (canceled)
 20. An isolated nucleic acid encoding SEQ ID NO.: 110.21. The isolated nucleic acid sequence of claim 20 that is SEQ ID NO.:111.
 22. An expression vector comprising the nucleic acid of claim 20.23. An expression vector comprising the nucleic acid of claim
 21. 24.The expression vector of claim 22 wherein the vector is a bacterial or aviral vector.
 25. The expression vector of claim 24 wherein the viralvector is selected from the group consisting of adenovirus, alphavirus,lentivirus, and poxvirus.
 26. The expression vector of claim 25 whereinpoxvirus is selected from the group consisting of vaccinia, fowlpox,avipox, orthopox, canary pox, swinepox, TROVAC, NYVAC, ALVAC(1),ALVAC(2), MVA, Wyeth and Poxvac-TC.
 27. The expression vector of claim25 wherein the poxvirus selected from the group consisting of NYVAC,ALVAC, and ALVAC(2).
 28. A method for inducing an immune response to atumor antigen in a mammal comprising administering a tumor antigen in afirst form directly into at least one lymph node of the mammal andsubsequently administering the tumor antigen in a second form differentfrom the first form directly into the at least one lymph node.
 29. Amethod according to claim 28 wherein the tumor antigen is selected fromthe group consisting of CEA, gp100, the MAGE family of proteins, DAGE,GAGE, RAGE, NY-ESO 1, Melan-A/MART 1, TRP-1, TRP-2, tyrosinase,HER-2/neu, MUC-1, p53, KSA, PSA, PSMA, fragments thereof and modifiedversions thereof.
 30. A method according to claim 28 wherein at leastone of said forms is a nucleic acid encoding the tumor antigen and thenucleic acid is selected from the group consisting of viral nucleic,acid, bacterial DNA, plasmid DNA, naked DNA, and RNA.
 31. A methodaccording to claim 30 wherein the viral nucleic acid is selected fromthe group consisting of adenoviral, alpha viral and poxviral nucleicacid.
 32. A method according to claim 31 wherein the poxviral nucleicacid selected from the group consisting of avipox, orthopox and suipoxnucleic acid.
 33. A method according to claim 31 wherein the poxviralnucleic acid is selected from the group consisting of vaccinia, fowlpox, canarypox and swinepox nucleic acid.
 34. A method according toclaim 31 wherein the poxviral nucleic acid is selected from the groupconsisting of MVA, NYVAC, TROVAC, and ALVAC nucleic acid.
 35. A methodaccording to claim 28 wherein the first form is a nucleic, acid and thesecond form is a peptide.